Focused Ultrasound-Induced Blood-Brain Barrier Opening Enhances GSK-3 Inhibitor Delivery for Amyloid-Beta Plaque Reduction

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease that is the leading cause of age-related dementia. Currently, therapeutic agent delivery to the CNS is a valued approach for AD therapy. Unfortunately, the CNS penetration is greatly hampered by the blood-brain barrier (BBB). Focused-ultrasound (FUS) has been demonstrated to temporally open the BBB, thus promoting therapeutic agent delivery to the CNS. Recently, the BBB opening procedure was further reported to clear the deposited Aβ plaque due to microglia activation. In this study, we aimed to evaluate whether the use of FUS-induced BBB opening to enhance GSK-3 inhibitor delivery, which would bring additive effect of Aβ plaque clearance by FUS with the reduction of Aβ plaque synthesis by GSK-3 inhibitor in an AD mice model. FUS-induced BBB opening on APPswe/PSEN1-dE9 transgenic mice was performed unilaterally, with the contralateral hemisphere serving as a reference. GSK-3 level was confirmed by immunohistochemistry (IHC) and autoradiography (ARG) was also conducted to quantitatively confirm the Aβ plaque reduction. Results from IHC showed GSK-3 inhibitor effectively reduced GSK-3 activity up to 61.3% with the addition of FUS-BBB opening and confirming the proposed therapeutic route. ARG also showed significant Aβ-plaque reduction up to 31.5%. This study reveals the therapeutic potentials of ultrasound to AD treatment, and may provide a useful strategy for neurodegenerative disease treatment.

Figure 1

The conceptual schematics of this study. The GSK-3 inhibitor was intraperitoneally (i.p.) injected and microbubbles were intravenously (i.v.) injected. During the sonication induced by a focused ultrasound transducer, GSK-3 inhibitor penetrated the blood-brain barrier to block/reduce the Aβ peptide synthesis and overexpression.

Figure 2

Assessment of GSK-3 distribution under various FUS and GSK-3 inhibitor treatment combinations. (A) Representative GSK-staining observed from wild type group (n = 3), control group (n = 3), FUS alone group (n = 6), GSK-3 inhibitor (AR) alone group (n = 6), combined treatment group (n = 9). (B) Quantitative analysis among groups in cortex (represented as the ratio of the GSK-3 immunoreactive area to the selected cortex region). Relative distribution was analyzed to 1.4 ± 0.07%, 1.14 ± 0.07%, 1.03 ± 0.06%, 0.48 ± 0.03%, and 0.21 ± 0.06% in wild type, control, FUS-alone, AR-alone, and AR + FUS group, respectively. AR alone groups showed significant inhibitions between control group (p < 0.001) and FUS-alone group (p < 0.001), whereas AR + FUS groups also showed significant differences between control group (p < 0.001), FUS-alone group (p < 0.001), and AR-alone group (p < 0.01). Control group showed a significant difference between the wild type group (p < 0.05). (C) Quantitative analysis among groups in hippocampus. The ratio of GSK-3 immunoreactive area to the whole hippocampus region showing the relative distribution of 0.45 ± 0.03%, 0.31 ± 0.02%, 0.29 ± 0.04%, 0.24 ± 0.04%, and 0.12 ± 0.01% in wild type, control, FUS-alone, AR-alone, and AR + FUS group, respectively. AR + FUS group showed significant differences between control group (p < 0.001), FUS alone (p < 0.01) as well as AR alone group (p < 0.05). Control group also showed a significant difference compared to the wild type group (p < 0.01). AR = AR-A014418; FUS = focused ultrasound. Scale bar = 500 μm.

Figure 3

Autoradiogram of brain section images for the different treatment groups. (A) GSK-3 inhibitor alone with wild type mice (n = 3); (B) FUS sonication alone with transgenic mice (n = 6); (C) GSK-3 inhibitor with transgenic mice (n = 6); (D) combination of GSK-3 inhibitor and FUS with transgenic mice (n = 9); and quantification of 4 groups with (E) cortex (F) hippocampus. AR-alone and FUS-alone groups didn’t show plaque differences when analyzing cortex and hippocampus regions. However, the plaque-reduction effect could be revealed in AR + FUS group as measured in cortex (31.5% reduction; 3099.9 ± 311.6 LAU/mm² vs. 4526.2 ± 574.9 LAU/mm²; p = 0.0401) or hippocampus (25.6% reduction; 1248.1 ± 136.9 LAU/mm² vs. 1678.1 ± 207.1 LAU/mm²; p = 0.0159). WT = wild type; AR = AR-A014418; FUS = focused ultrasound.

Figure 4

Assessment of amyloid-β plaques by fluorescent staining. (A) Representative Thioflavin-S stains among groups. (B) Quantification of amyloid-β plaque sizes in the cortex and (C) hippocampus, the data are represented as the ratio of the green fluorescence area from the region-of-interest (ROI). FUS alone only contribute a significant difference in hippocampus region when compared to the control group (15.1% reduction compared to control, p < 0.05). When GSK-3 inhibitor was administrated alone, significant reductions of amyloid-β plaque can be observed in both regions compared to the control group (31.6% and 22.6% reduction compared to control, with p < 0.05 and p < 0.01 in cortex and hippocampus respectively). When GSK-3 inhibitor was mediated by using FUS, there were reductive effects compared to respective control group (42.1% and 39.6% reduction compared to control, with p < 0.05 and p < 0.01 in cortex and hippocampus respectively). It was noted that combined AR + FUS treatment also induced the additive plaque reduction effect when compared to FUS alone (28.9%, p = 0.0003) and inhibitor alone (22%, p = 0.0236) in hippocampus when compared to control. AR = AR-A014418; FUS = focused ultrasound. Scale bar = 500 μm.